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Substrate parameters affecting propagation of juvenile freshwater pearl mussels margaritifera margaritifera (bivalvia: margaritiferidae)Interstitial habitat conditions are of critical importance to species inhabiting the hyporheic zone, particularly for moderately immobile species incapable of escaping poor habitat conditions. The critically endangered freshwater pearl mussel (Margaritifera margaritifera Linnaeus, 1758) has seen increasing propagation effort over the last three decades, often with mixed success. This study aimed to investigate parameters with the potential to affect juvenile survival in captivity by considering a range of habitat conditions within the substrate of a previously described propagation system using different substrate size classes (0.25–1 and 1–2mm) and cleaning regimes (weekly and monthly). Juvenile survival was highest in larger substrates, likely because of higher flow through larger pore spaces. This provided higher dissolved oxygen delivery in 1–2mm substrates cleaned weekly (8.26 ± 0.19 mg/L) and monthly (8.24 ± 0.44 mg/L), compared with 0.25–1mm substrates cleaned weekly (7.98 ± 0.44 mg/L) and monthly (6.78 ± 1.27 mg/L). The amount of organic material trapped in the substrate did not differ between treatments but the high concentrations of inorganic phosphorus liberated from ashed organic matter indicated phosphorus storage in phytoplankton. High dissolved oxygen concentrations and good water replacement between the water column and the substrate are crucial for survival in captive freshwater pearl mussels.